Breakout Question 1

1. Breakout Question 1 You are studying an uncharacterized membrane protein. The protein is found at the plasma membrane, but it is unclear how it interacts with the membrane as it has no hydrophobic domains. You perform FRAP under different conditions to study the protein’s lateral mobility and to characterize its association with the plasma membrane. • To assess the mobility of your protein, you construct a GFP-fusion protein and express it in cells. For your experiments, FRAP is performed after cells are exposed to the following experimental conditions: • Normal (no treatment) • Glycosylase treatment • Cholesterol extracted 2. Glycosylase treatment Phospholipids only 1. normal 3. Extract cholesterol Bleach Bleach Bleach Bleach Relative fluorescence Relative fluorescence Relative fluorescence Relative fluorescence A) What controls do you perform for your experiment? B) How does your protein interact with the plasma membrane? Time Time Time Time

2. Breakout Question 1 • Answer: • The labeled phospholipid serves as a control for a fast moving molecule, and the untreated group serves as a control for the baseline movement of the fusion protein. • Since the protein displays increased mobility following both glycosylase treatment and cholesterol extraction, it is most likely associated with a glyolipid or glycoprotein found within a lipid raft.

3. Breakout Question 2 You have reconstituted an in vitro nuclear transport system from frog oocyte nuclei, purified proteins and cytosolic extracts containing everything needed from the cytosol to support transport. You then use this system to examine what happens to nuclear transport under different conditions. For your experiment, you add a 100-fold excess of non-hydrolyzable GTP (GTPgS) to your system and examine transport. What do you think would happen? Include each student’s name and credit status on answer page

4. Answer Nuclear transport with halt because Ran-GTP will be replaced by Ran-GTPgS. This will lead to a depletion of Ran-GDP and a build-up of non-hydrolyzable Ran-GTPgS in the nucleus. The decrease in the pools of Ran-GDP will result in dampened import and less export as less nuclear Ran-GDP leads to less nuclear Ran-GTP necessary for export.

5. Breakout Question • You have a novel protein that contains a one putative ER signal sequence. To determine if the sequence functions to direct ER localization, you set up an in vitro translation experiment. You incubate increasing lengths of RNA encoding your protein with (+) and without (-) microsomes and determine the length of your protein product. • Does your protein contain a signal sequence? • If so, how much of the protein must be translated in order for recognition and cleavage by the peptidase?

6. Answer • the protein chain must be somewhere between 130 and 150 amino acids in length for the signal sequence to be fully accessible for cleavage. • Messenger RNA lengths vary by steps of 20 codons or 20 amino acids each in corresponding synthesized product. When translated in the absence or presence of microsomes, only mRNAs of 130 and 150 codons produce a product that displays any difference in size with the addition of microsomes. The 130-codon mRNA gives a product that is either full length—showing no difference in size when compared to the minus microsome product—or a smaller product that is the presumed result of signal peptidase cleavage. This suggests variable or incomplete accessibility of the product to signal peptidase. In contrast, the next-step-size-longer mRNA codes for product that is fully sensitive to signal peptidase cleavage when synthesized in the presence of microsomes. The key datum is the smaller size (faster mobility) of the product plus microsomes versus the product minus microsomes.

7. Breakout Question In a small molecule screen for modifiers of fibroblast cell motility, you identify a drug with unknown function. You hypothesize the drug might work by affecting actin dynamics. When motile cells were treated with drug X, movement was slowed. You perform in vitro actin polymerization assays to test the effect of drug X on F-actin dynamics. Shown are the results of performing the polymerization assays in the absence of drug X, in the presence of drug X alone, and in the presence of drug X and phalloidin, an F-actin stabilizing drug. Based on the results from this experiment, propose a mechanism for how drug X affects actin dynamics.

8. Answer Based on the data, drug X likely increases actin depolymerization as the effect of this drug is reversed by a chemical that stabilizes the filament.

9. Breakout question Microtubule (MT) dynamics involve sudden, rapid depolymerization events called catastrophes. These are followed by a rescue event, in which depolymerization stops and polymerization resumes. Dynamics at the plus end of a single, labeled MT were observed in an in vitro system containing cytosolic extracts, alpha and beta tubulin, and GTP. The results are shown in curve A. Curve B shows the changes in dynamics when a 10X excess of non-hydrolyzable GTP is added to the reaction. For what process in microtubule dynamics is GTP hydrolysis important? Use these data to explain the role of GTP hydrolysis in the balance between MT polymerization and depolymerization.

10. Answer • ATP hydrolysis occurs within the filament, and has the net overall effect of raising the critical concentration for G-Actin when ADP-bound monomers are at the end of the filament. Treadmilling describes net loss of G-Actin from the minus end and net gain of G-Actin at the plus end, roughly maintaining the length of the filament. This process can occur over a larger range of G-Actin when the minus end contains G-Actin-ADP and the plus end contains G-Actin-ATP.